Spinophilin as a novel regulator of M3R -mediated insulin release Recently, we tested the hypothesis that spinophilin (SPL), a multi-domain scaffolding protein, might play a role in modulating the activity of &#61538;eta-cell M3Rs. This study was prompted by the observation that SPL can regulate the activity of various CNS functions mediated by distinct GPCRs. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of BRET technology, suggested that SPL is able to recruit RGS4 to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins which act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. This is the first report demonstrating a role for SPL in regulating &#61538;eta-cell function. Given the importance of beta-cell M3Rs in maintaining normal blood glucose levels, our findings highlight the significant functional role of M3R (GPCR)-associated proteins in modulating &#61538;eta-cell biology. Such proteins may prove useful as targets for novel classes of drugs aimed at improving &#61538;eta-cell function for therapeutic purposes. Development of a designer GPCR useful for studying the roles of arrestins in regulating insulin release and other physiological functions Activated GPCRs are rapidly phosphorylated by GPCR kinases (GRKs), promoting interactions between the phosphorylated receptors and members of the arrestin protein family (arrestin-2 and -3), a process which interferes with receptor/G protein coupling. However, during the past decade, it has become increasingly clear that arrestin-2 and -3 can serve as adaptor proteins that transduce signals to multiple effector pathways. Although some progress has been made in delineating the physiological functions of arrestin signaling pathways in various tissues, much remains to be learned about the in vivo physiological relevance of arrestin-mediated signaling. Recently, various mutant muscarinic receptors have been developed that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs (alternative name: designer receptors exclusively activated by designer drug, DREADDs) have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. To explore the physiological relevance of arrestin-mediated signaling cascades, we recently developed a novel, arrestin-biased CNO-sensitive designer receptor (Rq(R165L)) as a novel experimental tool. This M3 receptor-based DREADD was no longer able to couple to G proteins but could recruit arrestins and promote ERK1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of MIN6 insulinoma cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.